Method and reagent system having a non-regenerative enzyme-coenzyme complex

ABSTRACT

The invention relates to a method and reagent system for detecting an analyte in a sample by means of an enzymatic reaction, involving the use of an enzyme-coenzyme complex as a stoichiometric reaction partner for the analyte present in the sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/EP03/05178 filed May 16, 2003 andclaims priority to foreign applications GERMANY 102-21-845.5 filed May16, 2002, GERMANY 102-21-804.4 filed May 16, 2002 andGERMANY102-21-846.3 filed Mar. 10, 2002.

The invention relates to a method and a reagent system for detecting ananalyte in a sample through an enzymatic reaction, comprising the use ofan enzyme-coenzyme complex as non-regenerable, in particularstoichiometric reactant for the analyte present in the sample.

The detection of analytes, for example glucose in blood, by enzymaticmethods is known. These entail the analyte to be determined beingbrought into contact with a suitable enzyme and a coenzyme, the enzymebeing employed in catalytic amounts. The redox equivalents produced onreduction or oxidation of the coenzyme are transferred to mediatorswhich are then detected electrochemically or photometrically in afurther step. A calibration provides a direct connection between themeasurement and the concentration of the analyte to be determined.

Sierra et al. (Anal. Chem. 69 (1997), 1471-1476) describe adetermination of blood glucose based on the intrinsic fluorescence ofglucose oxidase. In this method too, the enzyme is employed togetherwith its coenzyme FAD in catalytic amounts, with redox equivalents beingtransferred to oxygen as mediator.

Narayanaswamy et al. (Analytical Letters 21 (7) (1988), 1165-1175)describe a fluorescence measurement with glucose dehydrogenase and NADfor glucose determination. The enzyme is in this case employed incatalytic, i.e. non-stoichiometric, amounts. The fluorescencemeasurement detects the free NADH in the solution.

It is possible through the electrochemically active substances(mediators) required for the prior art detection systems to detect theanalytes to be determined only indirectly, i.e. via a plurality ofchemical reactions. For this purpose, a complicated adjustment of theconcentrations of the substances involved to optimize the reaction rateis often necessary. There is moreover the risk that the requiredelectrochemically active substances are unstable on prolonged storage.

The mediators often also have to be employed in large excess relative tothe enzyme-coenzyme system. The coenzyme has a high reactivity, so thatthe enzymic activity declines markedly on decomposition of the mediator,even in small amounts, e.g. <1% or on exposure to foreign substances,e.g. volatilization of the substances from packaging materials. This maylead to false signals in the determination of the analyte. Yet a furtherdisadvantage is that the determination times for the analytes arenormally in the region of at least a few seconds, for example forglucose in the region of >4 s, and the required sample volumes arelarge, e.g. >0.5 μl.

The object on which the present invention was based is at least partlyto avoid the described disadvantages of the prior art. It wasparticularly intended to provide a non-sensitive and rapid method forthe enzymatic detection of analytes, which leads to reliable measurementresults even in the absence of mediators or/and indicators.

This object is achieved by using an enzyme-coenzyme complex asstoichiometric reactant instead of, as usual, as catalyst. Detection ofthe analyte requires only a single reaction step and is thereforeextremely fast. The use of mediators and indicators, associated with theemployment of complex reaction mixtures, with low stability and highsusceptibility to interference, is no longer necessary.

One aspect of the invention is thus a method for detecting an analyte ina sample by an enzymatic reaction, comprising the steps:

-   -   (a) contacting the sample with a detection reagent comprising an        enzyme-coenzyme complex, where no regeneration of the coenzyme        takes place, and    -   (b) detecting a reaction of the analyte through a change in the        enzyme-coenzyme complex.

A further aspect of the invention is a reagent system for detecting ananalyte in a sample, comprising:

-   -   (a) a detection reagent comprising an enzyme-coenzyme complex,        where no regeneration of the coenzyme takes place, and    -   (b) a support to receive the detection reagent.

The present invention makes a simple qualitative or quantitativedetermination of analytes possible within a very short reaction time of,preferably, ≦5 s, particularly preferably ≦1 s, most preferably ≦0.1 s.The reaction is carried out under conditions with which no regenerationof the coenzyme takes place during the determination. It is moreoverpossible for a molecule enzyme-coenzyme complex to react only with asingle molecule of the analyte. The reaction is therefore expedientlycarried out in the absence of mediators or other substances able tobring about regeneration of the coenzyme.

The detection reagent comprises the enzyme-coenzyme complex in an amountsufficient to make qualitative or/and quantitative determination of theanalyte possible according to the desired test format. In particular,for quantitative determination of the analyte, the enzyme-coenzymecomplex is employed in an amount such that the number of reactingmolecules of the enzyme-coenzyme complex correlates with the analyteconcentration present in the sample. The enzyme-coenzyme complex isparticularly preferably employed in an at least stoichiometric amountrelative to the analyte present in the sample, preferably in astoichiometric excess relative to the analyte. In this connection, thestatement “in at least a stoichiometric amount” means that the size ofthe sample is adjusted relative to the number of molecules of theenzyme-coenzyme complex in such a way that, with the analyteconcentrations to be expected in the sample, the number of molecules ofthe enzyme-coenzyme complex which react with the analyte correlates withthe analyte concentration present in the sample. “Stoichiometric amount”preferably means that the number of molecules of the enzyme-coenzymecomplex corresponds to the maximum number of analyte molecules to beexpected in the investigative sample.

The method and the detection system permit the use of very small amountsof sample, for example sample volumes of ≦1 μl, in particular ≦0.1 μl.The sample can where appropriate also be diluted before contacting withthe detection reagent.

The method and detection system of the invention is suitable fordetermining any analytes, for example parameters in body fluids such as,for example, blood, serum, plasma or urine, but also in effluent samplesor foodstuffs. The method can also be carried out as wet test, e.g. in acuvette, or as dry test on an appropriate reagent support.

The analytes which can be determined are any biological or chemicalsubstances which are able to undergo a reaction, in particular a redoxreaction, with an enzyme-coenzyme complex, such as, for example,glucose, lactic acid, malic acid, glycerol, alcohol, cholesterol,triglycerides, ascorbic acid, cysteine, glutathione, peptides etc.

The enzymatic reaction is preferably a redox reaction in which thecoenzyme in the enzyme-coenzyme complex is reduced or oxidized. Theenzyme preferably used for a reaction of this type is an oxidoreductase.The enzyme particularly preferably used is a dehydrogenase, for exampleselected from a glucose dehydrogenase (E.C.1.1.1.47), lactatedehydrogenase (E.C.1.1.1.27, 1.1.1.28), malate dehydrogenase(E.C.1.1.1.37), glycerol dehydrogenase (E.C.1.1.1.6), alcoholdehydrogenase (E.C.1.1.1.1) or amino-acid dehydrogenase, e.g.L-amino-acid dehydrogenase (E.C.1.4.1.5). Further suitable enzymes areoxidases such as, for example, glucose oxidase (E.C.1.1.3.4) orcholesterol oxidase (E.C.1.1.3.6).

Coenzymes for the purposes of the present invention are preferablyorganic molecules which are linked covalently or noncovalently to anenzyme and are changed, for example oxidized or reduced, by theconversion of the analyte. Preferred examples of coenzymes are flavin,nicotine and quinone derivatives, for example flavin nucleosidederivatives such as, for example, FAD, FADH₂, FMN, FMNH₂, etc., nicotinenucleoside derivatives such as, for example, NAD⁺, NADH/H⁺, NADP⁺,NADPH/H⁺ etc. or ubiquinones such as, for example, coenzyme Q, PQQ etc.

The change in the coenzyme through reaction with the analyte can inprinciple be detected in any manner. It is possible in principle toemploy for this all methods known in the art for detecting enzymaticreactions. However, the change in the coenzyme is preferably detected byoptical methods. Optical detection methods include for example measuringabsorption, fluorescence, circular dichroism (CD), optical rotarydispersion (ORD), refractometry etc. The change in the coenzyme isparticularly preferably detected by measuring the fluorescence. Thefluorescence measurement is highly sensitive and makes it possible todetect even low concentrations of the analyte in miniaturized systems.

The method or detection system of the invention may comprise a liquidtest, in which case the reagent is present for example in the form of asolution or suspension in an aqueous or nonaqueous liquid or as powderor lyophilizate. However, the method and detection system of theinvention preferably comprises a dry test, in which case the reagent isapplied to a support. The support may comprise for example a test stripcomprising an absorbent or/and swellable material which is wetted by thesample liquid to be investigated.

In a particularly preferred embodiment, the detection reagent used is agel matrix with an enzyme-coenzyme complex embedded therein. The gelmatrix preferably has a layer thickness of ≦50 μm, in particular ≦5 μm,and is applied to a support, for example an at least partly opticallytransparent support. The gel matrix may be a matrix comprising one ormore soluble polymers, as in known dry test systems (e.g. AccuChekActive), and can be produced by knife application and drying. The matrixis preferably a polymer with a structure based on photopolymerizablesubstances such as, for example, acrylic monomers, e.g. acrylamideor/and acrylic esters such as polyethylene glycol diacrylate, orvinylaromatic monomers, e.g. 4-vinylbenzenesulfonic acid, orcombinations thereof. A gel matrix of this type can be produced byapplying a liquid which contains the reagent, comprising enzyme,photopolymerizable monomer and, where appropriate, coenzyme,photoinitiator or/and unreactive constituents, to an at least partlyoptically transparent support, for example to a plastics sheet, andirradiating, for example with UV light from the reverse side, so thatpolymerization of the monomer or of the monomers takes place on thesupport up to a predefined layer thickness. The layer thickness can becontrolled by adding absorbing substances to the reagent or/and throughthe duration or intensity of irradiation. Excess liquid reagent can beremoved and reused after the polymerization (see, for example, FIG. 2).

On the other hand, the gel matrix can also be produced by conventionalcoating procedures, in which case the liquid reagent is applied to asupport, brought to the desired thickness using suitable methods, e.g.using a knife, and then completely polymerized.

After inclusion by polymerization or embedding in the gel matrix, theenzyme is in a protected micro-environment. If the polymeric gel matrixis sufficiently crosslinked, the enzyme molecules are present in animmobilized form. Low molecular weight substances or glucose or otheranalytes or else coenzymes can, however, diffuse freely through thepolymer network.

The enzyme can either be included together with its coenzyme bypolymerization in the matrix or, after the polymerization, the matrixcan be brought into contact with a solution of the coenzyme, so that theappropriate enzyme-coenzyme complex is formed. The concentration of theenzyme in the gel matrix is preferably chosen to be high enough for astoichiometric reaction with the analyte to be determined, and a directdetermination of the coenzyme which is changed by the reaction, to bepossible. The reaction consists only of a single catalytic reaction, forexample, a redox reaction, which can take place in the region ofmilliseconds or microseconds. The coenzyme which is changed by thereaction is optimally protected from interfering influences throughbinding to the active center of the enzyme and, where appropriate,additionally by embedding in the gel matrix.

The invention is additionally to be explained by the following figuresand examples.

FIGURES

FIG. 1 shows a first embodiment of the detection system of theinvention. A reagent layer (2), e.g. a gel matrix with anenzyme-coenzyme complex, is applied to an optically transparent support(1). The enzyme-coenzyme complex is in a form such that no regenerationof the coenzyme can take place during the analyte determination. Asample (3), e.g. blood, is put on the reagent layer. Determination ofthe enzymatic reaction between the analyte contained in the sample (3),and the enzyme-coenzyme complex contained in the reagent layer (2) takesplace by optical methods. Light from a light source (4), e.g. a laser oran LED, is beamed from behind (through the support) onto the reagentlayer (2). Absorption light or fluorescent light beamed back from thesample is detected in a detector (5). Where appropriate—in particularfor detecting fluorescent light—an optical filter element (6) is put infront of the detector in order to block leakage of thefluorescence-exciting light.

FIG. 2 shows the production of a detection system of the invention. Aliquid reagent (12) is applied, for example in a first position (13), toan optically transparent support (11), e.g. a plastics sheet. The liquidreagent (12) is irradiated at a second position from below through thesupport (11) with light from a light source (14). At the same time, thesupport is moved in the direction (15) identified by the arrow. Apolymerized reagent layer (16) is formed directly on the support (11).Excess liquid reagent is present above the polymer layer (16). Thethickness of the polymerized reagent layer (16) can be controlledthrough the reagent composition, the duration and intensity of thebeaming in of light, and through the properties of the support (11).

FIG. 3 shows an embodiment of a fluorescence-based sensor from below. Apolymerized reagent layer, for example one produced by the continuousprocess in FIG. 2, can be cut and applied to a support (21) by use ofknown techniques. After application of the sample to the upper side,exciting light (23), e.g. UV light, is beamed in from a light sourcefrom below. The fluorescence (24), e.g. blue light, generated throughthe reaction of the analyte with the enzyme-coenzyme complex in thereagent layer (22) is detected with a detector.

It is also possible for a plurality of (identical or different) reagentsto be applied to a support. One example of such an embodiment in theform of a disk is shown in FIG. 4. A plurality of reagent spots (62) isdisposed on the optically transparent support (61).

FIGS. 5A and 5B show the fluorescence of a detection system of theinvention (glucose dehydrogenase and NAD⁺) with increasing glucoseconcentration under a CCD camera.

EXAMPLES Example 1 Stoichiometric Detection of Glucose in the GlucoseDehydrogenase (GlucDH)/NAD⁺ System in a Cuvette

100 mg/ml GlucDH are dissolved in buffer of pH 7 and mixed with theappropriate amount of NAD⁺. On addition of increasing amounts ofglucose, an increase in the fluorescence can be detected visually undera UV lamp (excitation wavelength 366 nm) (FIGS. 5A and 5B).

The solution with the enzyme system does not fluoresce without glucose.Nor do glucose and NAD⁺ result in any fluorescence.

Example 2 Detection of Glucose in the GlucDH/NAD⁺ System in a PolymerFilm

A suspension of the following substance was mixed in a plastic test tube

Formula 1 Amount Weight Substance [g] [%] Acrylamide 2.5 22.02Methylenebisacrylamide 0.7 6.17 2,2-Dimethoxy-2-phenylacetophenone 0.050.44 Glycerol 5 44.05 Hydroxyethyl methacrylate 1.4 12.33 Methylmethacrylate 0.4 3.52 Crodasinic O solution, pH 8, 0.3 g/1000 ml 1 8.81N,N′-(1,2-Dihydroxyethylene)bisacrylamide 0.3 2.64 TOTAL 11.35 100

0.5 ml of this suspension were mixed with 0.5 ml of a solution of GlucDH(100 mg/ml), and the mixture was homogenized free of air bubbles in anultrasonic bath.

The clear solution was poured onto a corona-treated polycarbonate sheet125 mm thick and illuminated with a conventional illumination apparatus(Isel UV illumination device 2) for 20 min. The sheet was briefly washedwith water and then dried in the air.

The resulting layer thickness was <2 μm A freshly prepared glucose/NAD⁺solution (GKL-3 solution, 300 mg/dl glucose, 1 ml/6.4 mg of NAD⁺) wasspotted on the film. A strong fluorescence was immediately visible undera UV lamp.

Example 3 Adding a UV Absorber to Influence the Layer Thickness

A polymer layer comprising a blue dye (absorption maximum ≈650 nm) forbetter identification was produced (formula 2). In a further experiment,a yellow dye was admixed as UV absorber to the initial formula (formula3).

Formula 2 Weight Substance Amount [%] Acrylamide  37.5 g 25.78 (0.53mol) Polyethylene glycol diacrylate,  52.5 g 36.10 Mw ≈ 575 g/mol (ca.0.96 mol) Solution of Crodasinic O (0.3 g/1 l)    50 g 34.384-Vinylbenzenesulfonic acid    5 g 3.442,2-Dimethoxy-2-phenylacetophenone   350 mg 0.24 photoinitiator Newmethylene blue N   100 mg 0.06 TOTAL 145.45 g 100

The mixture was homogenized by stirring and by ultrasonic bathtreatment, distributed with a pipette on a 140 μm Pokalon sheet(corona-treated, stage 4) and illuminated in a UV illumination device(Actina U4, W. Lemmen GmbH) for 1 min.

The resulting layer thickness was measured with a screw gage and was240.5 μm.

Formula 3 Substance Amount Weight [%] Formula 2    1 ml Ca. 99.99Mordant Yellow 7 (No. 686) 0.0001 g 0.001 (UV absorber) TOTAL Ca. 1001.0001 g

The mixture was distributed on a sheet and then polymerized as describedabove. The resulting layer thickness was measured with a screw gage andwas 79.3 μm.

This experiment shows that it is possible to influence the layerthickness. With reaction conditions which were otherwise the same, thelayer thickness without UV absorber is 240.5 μm (see above); only 79.3μm with UV absorber (Mordant Yellow 7).

1. A method for detecting an analyte in a sample by an enzymaticreaction, comprising the steps: (a) contacting the sample with adetection reagent comprising an enzyme-coenzyme complex, underconditions with which no regeneration of coenzyme takes place, wherebythe enzyme-coenzyme complex is employed in an at least stoichiometricamount relative to the analyte present in the sample, and (b) detectinga reaction of the analyte through a change in the enzyme-coenzymecomplex.
 2. The method as claimed in claim 1, characterized in that theenzymatic reaction comprises a redox reaction.
 3. The method as claimedin claim 1, characterized in that an oxidoreductase is used as enzyme,and a change in the coenzyme due to oxidation or reduction is detected.4. The method as claimed in claim 3, characterized in that the enzymeused is a dehydrogenase selected from a glucose dehydrogenase(E.C.1.1.1.47).
 5. The method as claimed in claim 1, characterized inthat a coenzyme selected from nicotine derivatives is used.
 6. Themethod as claimed in claim 5, characterized in that the coenzyme isNAD⁺.
 7. The method as claimed in claim 1, 2, 3, 4, 5 or 6,characterized in that the change in the coenzyme is detected by opticalmethods.
 8. The method as claimed in claim 7, characterized in that thechange in the coenzyme is detected by measuring absorption,fluorescence, circular dichroism, optical rotary dispersion orrefractometry.
 9. The method as claimed in claim 8, characterized inthat the change in the coenzyme is detected by measuring thefluorescence.
 10. The method as claimed in claim 1, characterized inthat a gel matrix with an enzyme-coenzyme complex embedded therein isused as detection reagent.
 11. The method as claimed in claim 10,characterized in that the gel matrix has a layer thickness of ≦50 μm.12. The method as claimed in claim 10 or 11, characterized in that a gelmatrix based on photo-polymerizable substances is used.
 13. The methodas claimed in claim 1, characterized in that an analyte in a body fluidis determined.
 14. The method as claimed in claim 13, characterized inthat a determination of glucose in blood is carried out.
 15. The methodas claimed in claim 1, characterized in that the duration of thereaction of the analyte is ≦5 s.
 16. The method as claimed in claim 1,characterized in that the reaction is carried out in the absence ofmediators able to react with the coenzyme.
 17. A method for detecting ananalyte in a sample by an enzymatic reaction, comprising the steps:contacting the sample with a detection reagent comprising a detectionreagent comprising an enzyme-coenzyme complex in a form such that noregeneration of the coenzyme takes place, whereby the enzyme-coenzymecomplex is employed in an at least stoichiometric amount relative to theanalyte present in the sample, and a support to receive the detectionreagent, under conditions with which no regeneration of coenzyme takesplace, and detecting a reaction of the analyte through a change in theenzyme-coenzyme complex.